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E Glover

Publications and source records attributed to E Glover.

At least 19 recordsLinked to original sources
PubMed2003

Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy.

Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations. The efficiency of this system to date has been disappointing, and therefore its utility for therapeutic application limited. We demonstrate here that efficiency and cellular specificity of trans-splicing is dependent on selection of the appropriate AAV serotype. Efficiency of transgene expression resulting from trans-splicing in skeletal muscle approaches that obtained when delivering the intact transgene when using AAV2 vectors packaged with AAV5 capsids (AAV2/5). This expands the potential of AAV vectors for retinal gene therapy. The use of AAV2/5 also increases the efficiency of trans-splicing in photoreceptors. Selection of the appropriate AAV serotype is likely to affect efficiency of trans-splicing in other organ systems as well.

Animals↗
PubMed2001

Identification of toxicologically predictive gene sets using cDNA microarrays.

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

Animals↗
PubMed2000

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice.

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Animals↗
PubMed2000

ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3, 7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress.

Animals↗
PubMed1999

Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina.

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV. CMV.EGFP. The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.

Animals↗
PubMed1994

Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species.

The mammalian aromatic hydrocarbon (Ah) receptor is a soluble protein involved in the regulation of gene expression by halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Little is known, however, about the presence and properties of this receptor in nonmammalian species. In these studies, we sought evidence for an Ah receptor in the liver or liver-equivalent of diverse species of invertebrate and vertebrate animals. Velocity sedimentation analysis of hepatic cytosol labeled with [3H]TCDD gave equivocal results with three species of marine fish. In subsequent studies, photoaffinity labeling with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin was used to identify the Ah receptor. Specific labeling (labeling that could be displaced by an excess of unlabeled ligand) was observed in seven species of teleost and elasmobranch fish, including winter flounder (Pleuronectes americanus), killifish (Fundulus heteroclitus), scup (Stenotomus chrysops), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and dogfish (Mustelus canis and Squalus acanthias). Specific labeling was also found in cytosolic fractions prepared from PLHC-1 fish hepatoma cells and livers of a turtle (Chrysemys picta) and a cetacean, the beluga whale Delphinapterus leucas. The fish Ah receptor was sensitive to conditions of tissue preparation; inclusion of proteinase inhibitors in the homogenization buffer stabilized the receptor in some species. There was heterogeneity in the apparent molecular mass of the largest specifically labeled band in each species; these ranged from 105 to 146 kDa, slightly larger on average than mammalian Ah receptors (95-130 kDa). In contrast to the results obtained with teleost and elasmobranch fish, no specifically labeled polypeptides were detectable in cytosol from two agnathan fish species (hagfish Myxine glutinosa and sea lamprey Petromyzon marinus), the tunicate Ciona intestinalis, or any of nine other invertebrate species representing eight classes in four phyla. Overall these results suggest that the Ah receptor evolved at least 450 million years ago, prior to the divergence of bony and cartilaginous fishes. Although the exact relationship between receptor presence and dioxin responsiveness in these species is uncertain, our data predict that the invertebrate species examined in this study, which appear to lack an Ah receptor protein like that seen in mammals and fish, may be less sensitive than vertebrates to the effects of environmental contaminants that act through this transcriptional regulator.

Affinity Labels↗
PubMed1994

Analysis of the four alleles of the murine aryl hydrocarbon receptor.

The cDNAs for the four murine aryl hydrocarbon (Ah) receptor alleles were cloned and sequenced, and the deduced amino acid sequences were compared. The Ahb-1 allele encodes a protein of 805 amino acids, the Ahd and Ahb-2 alleles encode proteins of 848 amino acids, and the Ahb-3 allele encodes a protein of 883 amino acids. The alleles differ by eight point mutations in the common open reading frame (the initial 805 amino acids) and by additional sequences at the carboxyl end. The amino halves of the proteins, containing a spliced leader sequence, a basic helix-loop-helix motif, and two 50-amino acid repeats (PAAS), have identical sequences except for a single amino acid change in the second PAAS box. The Ahd allele, which has a lower ligand binding affinity, differs from the Ahb-2 receptor by only two amino acids. Mutagenesis experiments with these cloned cDNAs, using in vitro transcription and translation and 2-[125I]iodo-7,8-dibromodibenzo-p-dioxin binding, indicate that the low ligand binding affinity of the Ahd allele is attributable to a valine at residue 375; changing this amino acid to an alanine, as in the Ahb-2 protein, enhances the affinity 4-fold. For in vitro translated Ahb-1 and Ahb-2 alleles the Kd values were approximately 6-10 pM and for Ahd the Kd value was approximately 37 pM. Using 5' truncation and mutations to produce 3' translation truncation sites, we mapped the ligand binding region for the Ahb-1 allele.

Alleles↗
PubMed1993

Physiological and genetic analyses of inbred mouse strains with a type I iodothyronine 5' deiodinase deficiency.

Inbred mouse strains differ in their capacity to deiodinate iododioxin and iodothyronines, with strains segregating into high or low activity groups. Metabolism of iododioxin occurs via the type I iodothyronine 5'deiodinase (5'DI), one of two enzymes that metabolize thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Recombinant inbred strains derived from crosses between high and low activity strains exhibit segregation characteristic of a single allele difference. Hepatic and renal 5'DI mRNA in a high (C57BL/6J) and low (C3H/HeJ) strain paralleled enzyme activity and concentration, in agreement with a recent report. 5'DI-deficient mice had twofold higher serum free T4 but normal free T3 and thyrotropin. Brown adipose tissue 5'DII was invariant between the two strains. Southern analyses using a 5'DI probe identified a restriction fragment length variant that segregated with 5'DI activity in 33 of 35 recombinant inbred strains derived from four different pairs of high and low activity parental strains. Recombination frequencies using previously mapped loci allowed assignment of the 5'DI gene to mouse chromosome 4 and identified its approximate chromosomal position. We propose the symbol Dio1 to denote the mouse 5'DI gene. Conserved linkage between this segment of mouse chromosome 4 and human HSA1p predicts this location for human Dio1.

Animals↗
PubMed1991

Purification and N-terminal amino acid sequence of the Ah receptor from the C57BL/6J mouse.

The Ah receptor is a presumed member of the superfamily of steroid/thyroid hormone receptors, a trace soluble protein present in a wide variety of vertebrate species that mediates the biological effects of halogenated aromatic hydrocarbons. In this paper, we report the purification to homogeneity of this protein (from the liver of C57BL/6J mice) and its N-terminal amino acid sequence. Selective covalent labeling of the Ah receptor in hepatic cytosol with the photoaffinity ligands 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin simplified identification and quantitation of the receptor and permitted purification under denaturing conditions. Photoaffinity-labeled hepatic cytosol was applied to a phosphocellulose column at 80 mM NaCl, and the fraction enriched with the Ah receptor eluted with 225 mM NaCl. The eluate was diluted to 150 mM NaCl and applied to a DEAE-cellulose column, and the enriched fraction eluted with 300 mM. These two ion exchange chromatography steps usually gave approximately 100-fold enrichment and 40-50% recovery of Ah receptor. The dilute protein in the eluate was precipitated with n-propanol/trichloroacetic acid and solubilized in formic acid. The sample was then subjected to three successive rounds of high performance liquid chromatography on C4 reverse phase columns. The final, shallow-gradient chromatography was able to resolve the unlabeled 95-kDa receptor protein from the later eluting 125I-photoaffinity-labeled protein. The pooled high performance liquid chromatography fractions subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels contained only the 95-kDa band upon staining with Coomassie blue R250 or silver. Using the above protocol, the Ah receptor was purified greater than 150,000-fold, to apparent homogeneity, with an overall yield of 3-5%. The N-terminal amino acid sequence of the purified peptide was determined to be ala/asp-ser-Arg-Lys-arg-Lys-Pro-Val-Gln-Lys-Thr-Val-Lys-Pro-Ile-Pro-Ala- Glu-Gly--Ile-Lys-ser-Asn-Pro-ser-Lys- (where the lowercase indicates a residue determined with less confidence).

Affinity Labels↗
PubMed1991

Characterization of polyclonal antibodies to the Ah receptor prepared by immunization with a synthetic peptide hapten.

A synthetic peptide based on the N-terminal amino acid sequence of the Ah receptor purified from C57BL/6J mice, linked to keyhole limpet hemocyanin, proved a remarkably good immunogen. All six rabbits that were immunized produced polyclonal antiserum that reacted with the synthetic peptide and the denatured and undenatured Ah receptor. Western blots were especially useful for antibody characterization; hepatic cytosol from C57BL/6J mice, in which the Ah receptor was photoaffinity labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin, was resolved by gel electrophoresis and electrotransferred to nitrocellulose. Co-incidence of the major band immunochemically stained with immunoaffinity-purified antibodies (with apparent Mr = 95,000) and the radiolabeled band on the autoradiograph indicated the specificity of the antibody. The estimated sensitivity of detection of the Ah receptor on a blot is 60 to 120 pg/200 micrograms of protein/gel lane. On Western blots, the antipeptide antibodies stained the photoaffinity-labeled Ah receptor from all four murine variants and all vertebrate forms examined (chicken, rodents, monkey, human), indicating conservation of these N-terminal epitopes. The immunoaffinity-purified antibodies also immunoprecipitated undenatured photoaffinity-labeled Ah receptor from diluted cytosol.

Affinity Labels↗
PubMed1990

Characterization and strain distribution pattern of the murine Ah receptor specified by the Ahd and Ahb-3 alleles.

Two allelic forms of the Ah receptor have been previously identified by covalent labeling of the hepatic cytosol fractions of inbred strains of mice with the photoaffinity ligand 2-azido-3-[125I] iodo-7,8-dibromodibenzo-p-dioxin and resolution of the labeled protein by denaturing gel electrophoresis: 1) a Mr 95,000 protein encoded by the Ahb-1 allele carried by the C57 and C58 family of mice, and 2) a Mr 104,000 protein encoded by the Ahb-2 allele present in other common inbred strains that are responsive to aromatic hydrocarbons (e.g., C3H/He, BALB/cBy, and A). In this report, 125I-photoaffinity labeling is used to characterize two further murine variants and the strains that carry them: 1) the low affinity Ah receptor (Ahd allele) in strains that are nonresponsive to aromatic hydrocarbons and 2) a newly identified, high affinity variant (Ahb-3) found in several strains recently derived from feral mice. The low affinity Ah receptor has been recently characterized by reversible ligand binding by Okey et al. [Mol. Pharmacol. 35:823-830 (1989)], through the inclusion of sodium molybdate in the buffers during tissue preparation and ligand incubation to stabilize the receptor. Examination of the Ah receptor in hepatic cytosol from 18 strains of mice carrying the Ahd allele, by preparation in molybdate and photoaffinity labeling, revealed that all strains express a Mr 104,000 protein. Tissue preparation in 20 mM sodium molybdate and subsequent dilution of the molybdate to approximately 0.5 mM during ligand incubation was found to enhance photoaffinity labeling of the high and low affinity allelic forms of the Ah receptor. A new variant of the receptor (Ahb-3) expressing a Mr 105,000 protein was detected in Mus molossinus, hortulanus, pahari, spretus, and caroli but was absent from the strains of Mus musculus or domesticus that were examined. Allelic variants were also distinguishable by thermolability, i.e., the half-life of specific ligand binding capacity upon incubation at 35 degrees. For the Ahb-1 allele (Mr 95,000) the t 1/2 (thermostability) is 20-30 min, for the Ahb-2 allele (Mr 104,000) the t 1/2 is 3-6 min, and for the Ahb-3 allele (Mr 105,000) the thermolability is intermediate.

Alleles↗
PubMed1989

[125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin-binding species in mouse liver induced by agonists for the Ah receptor: characterization and identification.

The admininistration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to C57BL/6J mice produces a dose-related increase in the hepatic uptake of [125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin ([125I]Cl3DpD) in vivo and the binding of the radioligand to liver homogenate in vitro [Mol. Pharmacol. 36: 121-127 (1989)]. The TCDD-induced hepatic binding species was found to be predominantly in the microsomal fraction and was inactivated by heating at 60 degree, trypsin, and mercurials. The TCDD-induced binding species was found to have an apparent equilibrium dissociation constant, KD, ([125I]Cl3DpD) of 56 +/- 16 nM and a pool size, Bmax, of 22 +/- 5 nmol/g of liver. A number of halogenated dibenzo-p-dioxins, biphenyls, and polycyclic aromatic hydrocarbons compete with [125I]Cl3DpD binding to this species; all are aromatic and planar. The distinctive profile of this binding species, a protein of large pool size induced in the microsomal fraction of liver but not other tissues and induced by agonists for the Ah receptor, suggested that this moiety might be cytochrome P3-450. The coincidence of the major microsomal species covalently labeled with the photoaffinity ligand [125I]2-iodo-3-azido-7,8-dibromodibenzo-p-dioxin and that immunochemically stained with polyclonal antiserum that binds to cytochrome P3-450 confirms this hypothesis. This is a novel role for a cytochrome P-450 isozyme, as an induced sequestration site that enhances the hepatic localization of the agonist drug.

Animals↗
PubMed1989

Stimulation of in vivo hepatic uptake and in vitro hepatic binding of [125I]2-lodo-3,7,8-trichlorodibenzo-p-dioxin by the administration of agonist for the Ah receptor.

[125I]2-lodo-3,7,8-trichlorodibenzo-p-dioxin ([125I]Cl3DpD), a radiolabeled, isosteric, analogue of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was synthesized and used to study in vivo tissue localization and in vivo tissue binding. Twenty-four hours after the administration of a tracer dose (1 X 10(-10) mol/kg) of [125I] Cl3DpD to C57BL/6J mice, the hepatic concentration of radioactivity was 1-2% of the administered dose, whereas in mice pretreated with TCDD (1 X 10(-7) mol/kg), the hepatic accumulation of radiolabel was 25-30% of that administered. Liver homogenate from TCDD-treated mice bound 4 to 10 times more [125I]Cl3DpD than homogenate from control mice. The enhancement of in vivo uptake and in vitro tissue binding of [125I]Cl3DpD by TCDD administration was confined to liver and was not observed in other tissues examined, kidney, lung, spleen, small intestines, and muscle. The administration of TCDD to C57BL/6J mice produces dose-related stimulation of in vivo hepatic uptake of [125I]Cl3DpD, binding of radioligand to liver homogenate, and hepatic aryl hydrocarbon hydroxylase activity, with the dose for half-maximal stimulation, ED50, varying from 1.5 to 4.0 x 10(-9) mol/kg. In congenic C57BL/6J (Ahd/Ahd) mice, which express the lower affinity Ah receptor, the ED50 values for all three responses were shifted to approximately 10-fold higher doses. 3,3',4,4',5,5'-Hexabromobiphenyl, a weak agonist for the Ah receptor produced a dose-related stimulation of these three responses in C57BL/6J mice (ED50 values of approximately 5 X 10(-7) mol/kg), but was without effect in C57BL/6J (Ahd/Ahd) mice. Stimulation of vivo hepatic uptake and in vitro liver homogenate binding of [125I]Cl3DpD was produced by administration of Ah agonists, such as 2,3,7,8-tetrachlorodibenzofuran and beta-naphthoflavone, but inactive congeners and other compounds that do not act via the Ah receptor, e.g. phenobarbital and pregnenolone-16 alpha-carbonitrile, did not evoke these effects. Thus, TCDD and other Ah agonists act through the Ah receptor to increase a liver binding species that increases the hepatic uptake of [125I]Cl3DpD in vivo and binding of this radioligand to liver homogenate in vitro.

Animals↗
PubMed1988

Ca2+-dependent proteolysis of the Ah receptor.

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.

Affinity Labels↗
PubMed1988

The development of a computerized assessment for minor psychiatric disorder.

This paper describes the development, validation and use of a computerized assessment for minor psychiatric disorder based on the Clinical Interview Schedule (CIS; Goldberg et al. 1970). There was good agreement between the computerized assessment and the CIS administered by psychiatrists, both in assessing overall severity and in defining 'cases' of psychiatric disorder. Individual symptoms elicited by the computer and the CIS were compared, and the levels of agreement found were similar to those from inter-observer studies of standardized interviews. Subjects from a variety of non-psychiatric settings regarded the assessment as acceptable, accurate and easy to use. It is concluded that this computerized assessment of neurotic symptoms is valid and reliable. It eliminates observer bias, it is an efficient use of research resources and it may have clinical applications in primary care.

Adolescent↗